A: The two main advantages of the T7-FlashScribe™ Transcription Kit V2 are improved RNA yields and a fast, 30-minute procedure.
A: We have produced 26-base RNA transcripts with the T7-FlashScribe™ Transcription Kit V2.
A: The longest transcription coding sequence transcribed using the T7-FlashScribe™ Transcription Kit V2 is approximately 11 kb.
A: The precipitate forms due to some kit components being formulated close to their solubility limits in solution. You may be able to salvage the buffer by heating to 37°C for a short period of time. The easiest way to avoid precipitation of reagents is to assemble the reaction at room temperature (22-25°C) and mix the reactions well prior to the 37°C or 42°C transcription incubation.
A: With lower concentrations of DNA template, you can increase RNA yields by increasing the reaction time from the recommended 30 minutes up to 2-4 hours, depending on the amount of template used. Increasing the reaction temperature from 37°C to 42°C also improves the yield.
A: The components of the reagents in a standard 20 μl reaction are very close to their solubility limits. If you set up the reaction on ice, some reaction components (such as the buffer and the nucleotides) will precipitate. After components precipitate, warming the reaction tube only partially resolubilizes the reagents. The T7-FlashScribe™ Enzyme Solution is added last, so it can be kept on ice until needed.
A: Yes, you can directly incorporate derivatized nucleotides (with moieties like Cy5, biotin, or digoxygenin) into the transcripts, or you can perform post-transcriptional labeling of purified RNA transcripts at the 5′ or 3′ ends. Please contact CELLSCRIPT™ for specific protocols.
A: Yes. While rarely done any more, radioactive probes may be made using post-transcriptional labeling. Generating radioactive RNA during the in vitro transcription reaction requires a lot of radioactive nucleotide, due to the high concentrations of radioactive NTPs required to prepare probes with high specific activity. This is extremely expensive and potentially dangerous. You can prepare radioactive RNA probes by using alkaline phosphatase to generate a 5′-hydroxyl end, followed by radioactive tagging using γ-32P-ATP and T4 Polynucleotide Kinase, or by using α-32P-(5′,3′)-bisphosphate NDPs and ligating to the 3′ end of the RNA using T4 RNA Ligase.
